The present invention relates to the field of cellular biology and more particularly, to a device and method for the study of cells. Specifically, the present invention is of a device including a picowell array for the study of cells as well as of a device configured for the study of cells that float in aqueous media.
Combinatorial methods in chemistry, cellular biology and biochemistry are essential for the near simultaneous preparation of multitudes of active entities such as molecules. Once such a multitude of molecules is prepared, it is necessary to study the effect of each one of the active entities on a living organism.
The study of the effects of stimuli such as exposure to active entities on living organisms is preferably initially performed on living cells. Since cell-functions include many interrelated pathways, cycles and chemical reactions, the study of an aggregate of cells, whether a homogenous or a heterogeneous aggregate, does not provide sufficiently detailed or interpretable results: rather a comprehensive study of the biological activity of an active entity may be advantageously performed by examining the effect of the active entity on a single isolated living cell. Thus, the use of single-cell assays is one of the most important tools for understanding biological systems and the influence thereupon of various stimuli such as exposure to active entities.
The combinatorial preparation of a multitudes of active entities coupled with the necessity of studying the effect of each one of the active entities on living organisms using single-cell assays, requires the development of high-throughput single live cell assays. There is a need for the study of real-time responses to stimuli in large and heterogeneous cell populations at an individual cell level. In such studies it is essential to have the ability to define multiple characteristics of each individual cell, as well as the individual cell response to the experimental stimulus of interest.
In the art, various methods and devices for studying living cells are known.
One method of studying cells involves placing cells on a bottom surface of a vessel and observing the behavior of the cell in response to stimuli. Typically used vessels include slides with recesses and Petri dishes. To allow for simultaneous study of distinct groups of cells exposed to similar or different stimuli, multiwell plates are most commonly used. A multiwell plates is substantially a group of standard sized individual vessels physically associated in a standard way allowing for simplified simultaneous or sequential studies of separated groups of cells. Multiwell plates having 6, 12, 24, 48, 96, 384 or even 1536 wells on a standard ca. 8.5 cm by ca. 12.5 cm footprint are well known in the art. Such multiwell plates are provided with an 2n by 3n array of rectangular packed wells. The diameter of the wells of a plate depends on the number of wells and is generally greater than about 250 microns (for a 1536 well plate). The volume of the wells depends on the number of wells and the depth thereof but generally is greater than 5×10−6 liter (for a 1536 well plate). The standardization of the multiwell plate format is a great advantage for researchers, allowing the use of standardized products including robotic handling devices, automated sample handlers, sample dispensers, plate readers, observation components, plate washers, software and such accessories as multifilters.
When a vessel having a planar bottom surface is used to study cells, the cells are most often studied as a group having an aggregate of properties of the individual cells. Since the cells are studied as a group, in such studies the identity of individual cells is not important. Such studies are of limited utility due to the fact that naturally occurring cell populations are rarely homogenous and often it is the heterogenity and the differences of behavior of cells that is of interest.
Efforts have been made to use vessels having a planar bottom surface to study cells as individuals but such efforts are plagued with many difficulties. A first difficulty is that cells have a tendency to clump together in variably sized groups at random locations, and often stack one on top of the other. The clumping and stacking of cells together makes it virtually impossible to delineate the borders of one cell from another, see discussion in unpublished PCT Patent Application No. IL2005/000719 of the inventor, rendering it virtually impossible to identify which cell has a given behavior. Further, the fact that cells are randomly distributed over a featureless surface coupled to the fact that cells often move due to currents makes it virtually impossible to definitely identify a specific cell without continuous observation of the cell. These factors further render high-throughput imaging (for example for morphological studies) challenging as acquiring an individual cell and focusing thereon is extremely difficult. Such variability in location also makes high-throughput signal processing (for example, detection of light emitted by a single cell through fluorescent processes) challenging as light must be gathered from the entire area of the well, decreasing the signal to noise ratio. Further, a cell held in a well of a multiwell plate well can be physically or chemically manipulated (for example, isolation or movement of a single selected cell or single type of cell, changing media or introducing active entities) only with difficulty. Further, addition of a reagent to a well or even the slightest incidental jostling of the vessel causes cells held therein to move randomly leading to the loss of identity of individual cells and rendering experiments difficult to perform, limited in scope and slow. Further, cell behavior is influenced by contact with other cells. If, to avoid the above difficulties, a multiwell plate holds only one cell per well, loading of the plate is very low (about 1536 cells in 65 cm2, or 24 cells cm−2). Thus, flat-bottomed vessels are in general only suitable for the study of homogenous or heterogenous aggregates of cells as a group.
Flat-bottomed vessels are also unsuitable for the study of cells undergoing apoptosis. It is known to study biological processes by exposing a monolayer of cells adhering to the bottom of a flat-bottomed vessel to a stimulus that initiates apoptosis. Once a cell begins apoptosis, the adhesion of the cell to the bottom of the vessel is no longer sufficient: the cell detaches from the bottom and is carried away by incidental fluid currents in the vessel. The cell is no longer observable and its identity lost.
Flat-bottomed vessels are also unsuitable for the study of non-adhering cells. Just as cells undergoing apoptosis, in flat-bottomed vessels non-adhering cells can be studied as individuals only with difficulty. This is a major disadvantage as non-adhering cells are crucial for research in drug discovery, stem cell therapy, cancer and immunological diseases detection, diagnosis and therapy. For example, blood contains seven heterogeneous types of non-adherent cells, all which perform essential functions, from carrying oxygen to providing immunity against disease.
It is known to study cells in a vessel having a planar bottom surface provided with an array of cell-localizing features. In such vessels, each cell is held in a specific location that is individually addressable allowing the identity of a given cell to be retained even without continuous observation, see for example Mrksich and Whitesides, Ann. Rev. Biophys. Biomol. Struct. 1996, 25, 55-78; Craighead et al., J. Vac. Sci. Technol. 1982, 20, 316; Singhvi et al., Science 1994, 264, 696-698; Aplin and Hughes, Analyt. Biochem. 1981, 113, 144-148, U.S. Pat. No. 4,729,949, U.S. Pat. No. 5,324,591, U.S. Pat. No. 6,103,479 and PCT Patent Application No. US99/04473 published as WO 99/45357. Many such devices deform the shape of the cells or require cell binding or adhesion to a vessel surface, adversely effecting the results of performed studies
In PCT patent applications IL2001/00992 published as WO2003/035824 (“Interactive transparent individual cells biochip processor”), IL2004/000571 published as WO2004/113492 (“Improved materials for constructing cell-chips, cell-chip covers, cell-chip coats, processed cell-chips and uses thereof”), IL2004/000194 published as WO2004/077009 (“A method and device for manipulating individual small objects”), IL2004/000661 published as WO2005/007796 (“Improved multiwell plate”), and unpublished IL2005/000801 (“Device for the study of cells”) all of the inventor or applicant, are provided picowell-bearing devices. A picowell-bearing device is a device for the study of cells that has at least one picowell-bearing component. A picowell-bearing component is a component having at least one, but generally a plurality of picowells, each picowell configured to hold at least one cell. In such devices, individual cells are generally held in a substantially natural state, preferably unadhered, in individual adressable picowells, preferably each cell alone in an individual picowell. In the art, a picowell-bearing component of a picowell-bearing device is often a chip, a plate or other substantially planar component. Such a component is termed a “carrier”.
A picowell, as the name suggests, is small well-shaped feature (including cavities, dimples, depressions, tubes and enclosures) configured to localize cells in well-defined locations on the bottom surface of a vessel. Since cells range in size from about 1 microns to about 100 (or even more) microns diameter there is no single picowell size that is appropriate for holding a single cell of any type. That said, the dimensions of the typical individual picowell in the picowell-bearing components known in the art have dimensions of between about 1 microns up to about 200 microns or even up to about 250 microns, depending on the exact implementation. For example, a device designed for the study of single isolated 20 micron cells typically has picowells of dimensions of about 20 microns. In other cases, larger picowells are used to study the interactions of a few cells held together in one picowell. For example, a 200 or even 250 micron picowell is recognized as being useful for the study of the interactions of two or three cells, see PCT Patent Application No. IL01/00992 published as WO 03/035824.
A feature that increases the utility of picowell-bearing devices is that each individual picowell is individually addressable. By individual addressability is meant that each picowell is identifiable from amongst the other picowells of a picowell array allowing each picowell to be registered, found, observed or studied as desired. For example, while cells are held in picowells of a picowell-bearing component, each cell is characterized and the respective picowell wherein that cell is held is noted. When desired, the observation component of the picowell-bearing device is directed to observe the picowell where a specific cell is held. One method of implementing individual addressability is by the use of fiducial points or other features (such as signs or labels), generally on the picowell-bearing component. Another method of implementing individual addressability is by arranging the picowells in a picowell-array and finding a specific picowell by counting. Another method of implementing individual addressability is by providing a dedicated observation component for each picowell. Another method is by observing a whole picowell array substantially in its entirety.
An additional feature that increases the utility of picowell-bearing devices is that the picowells of the picowell array are close together, preferably being juxtaposed, that is, the area occupied by a picowell-array is substantially entirely made up of picowells with little or no inter picowell area, see FIG. 1, a reproduction of a photograph of a picowell-array from above, such as described in PCT Patent Application No. IL01/00992. In FIG. 1 is seen a part of a hexagonally packed picowell array 10 including a plurality of hexagonal picowells 12, some holding living cells 14. It is seen that the inter picowell areas 16 make up only a minor percentage of the total area of picowell-array 10. Preferably the wells are knife-edged, that is the interwell surfaces are substantially non-existent. Such a feature allows simple loading of cells by sedimentation, allows very high-loading (expressed in units of cells per unit area), avoids stacking of cells, and clearly separates and prevents physical contact between cells held in adjacent picowells 12.
An additional feature that increases the utility of embodiments of picowell-bearing devices is the use of optical properties of the picowell array such as of the picowell bottom surface or picowell walls for automatic delineation of picowell borders in an image of the picowell array, allowing high throughput image acquisition, image processing and experimental analysis as described in PCT patent application IL2005/000719 published as WO2006/003664 of the inventor.
The use of the picowell-bearing devices described above allows large number of cells to be studied as individuals. In embodiments of picowell-bearing devices such as described in PCT patent applications IL2001/00992 published as WO2003/035824, complex experiments involving serial addition of reagents and the like are performed with dedicated microfluidics systems including flow generators such as pumps or syringes. In embodiments described therein, a picowell array is located in a substantially sealed chamber, the chamber in fluid communication with at least one fluid inlet and at least one fluid outlet. In embodiments, cells are loaded onto the picowell array by sedimentation: a moveable wall of the chamber in which the picowell array is located is moved, a solution of cells is introduced through the consequent opening, the cells allowed to settle in the picowells, and the moveable wall replaced so as to reseal the chamber.
Despite the unparalleled utility of such picowell-bearing devices, such devices have a number of limitations. A first limitation is that the devices are generally difficult to use by an unskilled person and are not completely suitable for integration with a robotics system for automatised use. A second limitation is that the devices are relatively complex to assemble. A third limitation is that the flow rate of liquids through picowell-array holding chamber is relatively limited. If a flow rate is too high, pressure in the chamber increases to the point where the lid is lifted upwards, allowing leakage of liquid from under the lid, or even detached. In embodiments this limitation is overcome by permanently securing all chamber walls, for example by using an adhesive so that no wall is moveable. In such embodiments, cells are loaded onto the picowell array through a fluid inlet, a time consuming step that is less efficient than loading of the picowell array by cell sedimentation.
Many of the above limitations are overcome by the teachings of unpublished PCT patent application IL2005/000801 of the inventor. In embodiments of the device taught therein, a picowell array is located inside a depression on a plateau, the plateau provided with a moveable wall that is substantially a lid, the space between the lid and the plateau defining a capillary channel. For use, cells are loaded by sedimentation when the lid is open and the lid subsequently shut. Liquid added at one end of the lid is transported past the picowell array through the capillary channel defined between the lid and the plateau. Embodiments of the device are remarkably simple to manufacture and simple for use, even by unskilled practitioners. A disadvantage of embodiments of such devices is that the flow rate and total volume of liquid that can be added is limited.
An additional disadvantage of the devices known in the art including the discussed above is that the devices are not exceptionally suited for the studies of cells, such as adipocytes, that have a density lower than the aqueous medium in which the cells are found and therefore float.
It would be highly advantageous to have a device for the study of cells not having at least some of the disadvantages of the prior art.